Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Meningeal macrophages inhibit chemokine signaling in pre-tumor cells to suppress mouse medulloblastoma initiation.

The microenvironment profoundly influences tumor initiation across numerous tissues but remains understudied in brain tumors. In the cerebellum, canonical Wnt signaling controlled by Norrin/Frizzled4 (Fzd4) activation in meningeal endothelial cells is a potent inhibitor of preneoplasia and tumor progression in mouse models of Sonic hedgehog medulloblastoma (Shh-MB). Single-cell transcriptome profiling and phenotyping of the meninges indicate that Norrin/Frizzled4 sustains the activation of meningeal macrophages (mMΦs), characterized by Lyve1 and CXCL4 expression, during the critical preneoplastic period. Depleting mMΦs during this period enhances preneoplasia and tumorigenesis, phenocopying the effects of Norrin loss. The anti-tumorigenic function of mMΦs is derived from the expression of CXCL4, which counters CXCL12/CXCR4 signaling in pre-tumor cells, thereby inhibiting cell-cycle progression and promoting migration away from the pre-tumor niche. These findings identify a pivotal role for mMΦs as key mediators in chemokine-regulated anti-cancer crosstalk between the stroma and pre-tumor cells in the control of MB initiation.

Three dimensional reconstruction of the mouse cerebellum in Hedgehog-driven medulloblastoma models to identify Norrin-dependent effects on preneoplasia.

Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/- mice and EGL hyperplasia in Neurod2-SmoA1+/- mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.

Flow cytometry detection and quantification of CAR T cells into solid tumors.

Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation.

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.